The capabilities of electron microscopy, principally of high-resolution transmission electron microscope, are considerable. Thanks to this technology, it has been possible to make great strides in research and information recovery in the context of examination of, for example, biological ultrastructures or semiconductor structures.
Because of the high vacuum that exists in the transmission electron microscope (TEM) and the high-energy electron beam, a sample preparation process that retains the structure is usually necessary. This is especially the case with biological samples. For high-resolution transmission electron microscope imaging, it is furthermore absolutely necessary that the sample be sufficiently thin. TEM samples are applied onto suitable sample carriers for examination. These are typically very small, round, delicate grids with a diameter from 2 to 3 mm. The grids have variously shaped holes (honeycomb, slits, etc.) or a lattice of a defined mesh count. The grids are usually coated with a thin film and can moreover also comprise further coatings.
For examination under the electron microscope, the sample carrier with the sample located on it must be retained in a suitable specimen holder apparatus. For most applications, the specimen holder apparatus is implemented as a goniometer, side-entry goniometers being predominantly used. With one-piece specimen holder apparatuses, the sample carrier is placed in an aperture of the sample holder apparatus and retained there. Alternatively, multi-part specimen holder apparatuses such as those described in EP 1 868 225 A1 and EP 1 947 675 A1 are increasingly be used. With these multi-part apparatuses, the sample carrier is first retained in a frame-like mount (also referred to as a “cartridge”), and the mount is then reversibly fastened in a corresponding holder of the specimen holder apparatus.
Electron microscope specimen holder apparatuses and mount apparatuses must meet specific requirements.
In addition to very high mechanical stability and high-vacuum compatibility, the retention and mounting of a sample carrier in the specimen holder apparatus is highly relevant because of the delicate nature of the sample carriers that are used. For proper examination and in order to prevent loss of the sample carrier, the sample carrier must be retained in a stable and vibration-free manner. In addition, distortions of the delicate sample carrier must be avoided, since otherwise it is easily damaged. In known specimen holder apparatuses such as those that have been disclosed, for example, by EP 1 947 675 A1 and U.S. Pat. No. 6,002,136, the grid is held in the aperture of the specimen holder apparatus with the aid of a securing ring. This securing ring is usually press-fitted using a tool, which has a disadvantageous effect on the coating of the sample carrier. These coatings are typically very brittle and can easily be destroyed by distortion upon installation of the sample carrier and the securing ring. Furthermore, the securing rings are small elements that are cumbersome to handle and can easily become lost, especially when they need to be loaded into the installation tool.
For many applications, provision is made for highly precise and stable tilting of the specimen around one or more axes lying in the specimen plane. WO 00/10191, for example, describes a side-entry specimen holder apparatus with double tilting. With the construction described above using a securing ring, the potential of such applications can, disadvantageously, not be entirely exploited. Securing rings require a groove in order to press the sample carrier onto the annular support in the aperture. The securing ring and the groove necessitate an annular configuration that is slightly smaller than the grid diameter. This results in a limitation of the tilt angle, and consequently in reduced recovery of information from electron microscopy observation.
For certain electron microscope applications it is furthermore necessary for the sample to be transferable from the sample preparation apparatus into the TEM, and for good thermal contact to exist between the EM specimen holder apparatuses and the sample. This is essential especially in the case of cryo-electron microscopic applications in structural biology. With this technology, an aqueous sample is cryofixed, i.e. it is cooled very rapidly, avoiding the formation of ice crystals. The objects to be examined, for example cells, enzymes, viruses, or lipid layers, are thereby embedded in a thin, vitrified ice layer. Transfer of the cryofixed sample involves critical handling and possibilities for contamination. Specially cooled EM specimen holder apparatuses and mounts, which enable the transfer of a cryofixed sample, are used for this.